Confocal Microscopy: Adding the Third Dimension
Bi/BE 177: Principles of
Modern Microscopy
Lecture 07: Confocal Microscopy. Adding the Third Dimension
Andres Collazo, Director Biological Imaging Facility
Ke Ding, Graduate Student, TA
Wan-Rong (Sandy) Wong, Graduate Student, TA
Lecture 7: Confocal Microscopy
•
Optical Sectioning: adding the third dimension
•
Wide-field Imaging
•
Point Spread Function
•
Deconvolution
•
Confocal Laser Scanning Microscopy
•
Confocal Aperture
•
Optical aberrations
•
Spinning disk confocal
•
Two-photon Laser Scanning Microscopy
•
Fluorescent proteins
Questions about last lecture?
Improve fluorescence with optical
sectioning
•
Wide-field microscopy
•
Illuminating whole field of
view
•
Confocal microscopy
•
Spot scanning
•
Near-field microscopy
•
For super-resolution
•
TIRF
•
Remember, typical
compound microscope is
not 3D, even though
binocular
Overview of Optical sectioning Methods
•
Deconvolution
•
Point-Spread function (PSF) information is used to calculate light back to its origin
•
Post processing of an image stack
•
Confocal and Multi-photon Laser Scanning Microscopy
•
Pinhole prevents out-of-focus light getting to the sensor(s) (PMT - Photomultiplier)
•
Multi Photon does not require pinhole
•
Spinning disk systems
•
A large number of pinholes (used for excitation and emission) is used to prevent out-of-
focus light getting to the camera
•
Especially those using Nipkow disk and microlens
•
Structured Illumination Microscopy (SIM)
•
Light sheet fluorescence microscopy, also called selective/single plane
illumination microscopy (SPIM)
Widefield imaging: entire field of view illuminated
And projected onto a planar sensor
Relationship between diffraction, airy disk and point
spread function
•
Airy disk – 2D
•
Point spread function -3D
•
Though often defined as
the same that is not quite
true
Point Spread Function is three dimensional
Subdiffraction limit spot
Image of subdiffraction limit spot
Thus, each spot in specimen will be blurred onto the sensor
(Aperture and
“
Missing Cone
”
)
https://micro.magnet.fsu.edu/primer/java/imageformation/depthoffield/index.html
To reduce contribution of blurring to the image: Deconvolution
Image blurred by PSF
Compute model of what might
have generated the image
Compute how model
would be blurred by
PSF
Compare
and iterate
Deconvolution depends on data from focal planes above and below
focal plane being analyzed.
Image deconvolution
•
Inputs:
•
3-D image stack
•
3-D PSF (bead image)
•
Requires:
•
Time
•
Computer memory
•
Artifacts?
•
Algorithms so good now
Note: z-axis blurring from the missing cone is minimized
but not eliminated
A
Optical Sectioning even when 3D image stack is
incomplete
•
Deconvolution
•
Confocal microscopy
Top: Macrophage -
tubulin,
actin
&
nucleus
.
Bottom: Imaginal disc –
α
-tubulin
,
γ
-tubulin
.
Optical Sectioning: Increased Contrast and Sharpness.
Examples: Zebrafish images, Inner ear
Zebrafish wide-field, optical section
Confocal microscope Z-stack
PMT
Detector
Detection
Pinhole
Excitation
Pinhole
Excitation
Laser
Objective
Dichroic
Beam
Splitter
C
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j
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a
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e
F
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c
a
l
P
l
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e
s
How else to fill in the missing cone?
Need more data in the Z-axis --> Confocal microscopy
C
o
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f
o
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p
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h
o
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e
s
www.olympusfluoview.com
Confocal Microscopy just a form of
Fluorescence Microscopy
Three
confocal
places
Confocal Microscopy (Minsky, 1957)
•
Yes that Marvin Minsky of MIT AI (Artificial
Intelligence) lab fame.
Focal Points
Identical Lens
Pinhole: Axial Filtering
But at a cost in brightness:
•
Thinner section means less labeled material in image
•
Aperture rejects some in focus light
•
Subtle scattering or distortion rejects more light
Aperture trims the PSF: increased resolution in XY plane
How N.A. and air disk effects resolution
Resolution, Signal and Pinhole Diameter
http://depts.washington.edu/keck/leica/pinhole.htm
Best Resolution
Best Signal to Noise
Light projected on a
single spot in the
specimen
Good: excitation falls
off by the distance from
the focus squared
Spatial filter in front of
the detector
Good: detection falls
off by the distance from
the focus squared
Bad: illumination of
regions that are not
used to generate an
image
Optical sectioning
Combined, sensitivity falls off by (distance from the focus)
4
Why does confocal add depth discrimination?
•
Only a single point is
imaged at a time.
•
Detector signal must be
decoded by a computer
to reconstruct image.
•
Imaging point needs to
be scanned somehow.
But this arrangement generates an “image” of
only one point in the specimen
Scan Specimen
Good:
•
Microscope works on axis
•
Best correction for optical
aberrations
•
Most uniform light
collection efficiency
Bad:
•
Slow
•
Sloshes specimen
Scan Microscope Head
Good:
•
Specimen doesn’
t move
•
Microscope works on axis
•
Best correction for optical
aberrations
•
Most uniform light
collection efficiency
Bad:
•
Slow
•
Optics can be more
complicated
Scan Laser
Good:
•
Faster
•
Specimen moves slowly—
less sloshing
Bad:
•
Very high requirements on
objective
•
Light collection may be
non-uniform off-axis
•
More complicated
Confocal Terminology
•
LSCM
•
Laser Scanning Confocal Microscopy
•
CLSM
•
Confocal Laser Scanning Microscopy
•
CSLM
•
Confocal Scanning Laser Microscopy
•
LSM
•
Laser Scanning Microscopy
Optical Aberrations: Imperfections in optical
systems
•
Spherical
•
Chromatic
(blue=shorter
wavelength)
•
Curvature of field
Spherical Aberration
Spherical aberration: Light misses aperture (and defocused)
f
o
i
Higher index of refraction results in shorter f
•
Chromatic Aberration
•
Lateral (magnification)
•
Axial (focus shift)
Lateral chromatic aberration - light misses aperture
Detector
f
o
i
Results in a
“
port hole
”
image: dimmer at edges
Curvature of field: Flat object does not project
a flat image
Optical Aberrations:
•
Image dimmer with depth
•
Image dimmer at edges
•
Image resolution compromised
Can’
t fight losses with smaller NA
Remember N.A. and image brightness
Epifluorescence
Brightness = fn (NA
4
/ magnification
2
)
10x 0.5 NA is 8 times brighter than 10x 0.3NA
Optical Aberrations:
•
Image dimmer with depth
•
Image dimmer at edges
•
Image resolution compromised
Can’
t fight losses with smaller NA
NA also has a major effect on image resolution
Minimum resolvable distance
d
min
= 1.22
/ (NA
objective
+NA
condenser
)
Larger N.A. can collect higher order rays
Best way to deal with Aberrations is to have high
performance objective
•
High Numerical Aperture
•
Water immersion for live cell
imaging
•
Correction for spherical
aberrations
•
Flat field correction
•
Chromatically corrected
over many different
wavelengths
•
Transmit UV and IR
•
Two photon
•
All light travels through the same zone
•
Angle at which the light travels dictates
the position in the specimen plane
•
Not imaging but illumination conjugate
plane.
How to scan the laser beam?
Place galvanometer mirror at the telecentric point
laser
How to scan the laser beam?
Place galvanometer mirror at the telecentric point
Modern closed-loop
galvanometer-driven laser
scanning mirror from Scanlab
If not at telecentric point,
Spherical aberration results
How can two mirrors be at the
same point??
laser
Position is critical
Place galvanometer mirror at the telecentric point
If not at telecentric point,
Spherical aberration results
How can two mirrors be at the
same point??
Optical relay
(without aberration)
laser
Scanners can introduce optical aberrations
Place galvanometer mirror at the telecentric point
Problem: Optical aberrations from simple lens
systems
f
o
i
Focal
Point
Focal
Point
f
Simple pair of lenses can minimize problem
(equal and opposite distortions)
Focal
Point
f
1:1 Image relay
Optically two mirrors can be at
the same point
Optical relay
(without aberration)
Position is critical
P
l
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Limitations: Phototoxicity
•
Sample is continuously exposed to light.
•
Weaker signal within sample requires stronger
excitation and causes more toxicity.
Limitations: Photobleaching
•
Scanning causes repeated exposure above and below.
Loss of sectioning by Scattering
How else to do confocal microscopy?
Confocal microscopes can be slow. Can we go faster?
Illumination
through this side
Alignment is critical
Most of light hits mask not hole
Tandem spinning disk scanner
EMCCD
or CMOS
Camera
Detection
through this side
Laser
~1% pass
Nipkow disk
>>1% pass
Yokogawa
Nipkow disk with microlenses
http://zeiss-campus.magnet.fsu.edu/tutorials/spinningdisk/yokogawa/index.html
Nipkow disk with microlenses
Optical sectioning without an aperture?
Two-Photon laser-scanning microscopy
4nsec
0.8 emitted
Conventional Fluorescence
(Jablonski diagram)
Emitted light is a
linear function of the
exciting light
4nsec
0.8 emitted
Excitation from coincident
absorption of two photons
Two-Photon Excited Fluorescence
(Jablonski diagram)
Very low probability: required intense pulsed laser light
Requires two photons: emission is a function of (exciting light)
2
Exciting light falls off by (distance from focus)
2
Thus, Emission falls off by (distance from focus)
4
--> Optical Sectioning without a confocal aperture!!
Two-Photon Excited Fluorescence
Optical sectioning by non-linear absorbance
--> broad excitation maxima
Two-Photon microscopy
TPLSM excitation at 900nm excites multiple dyes and GFP variants
Two-photon microscopy is somewhat color-blind
Two Photon Microscopy
Advantages
•
No need for pinhole
•
No bleaching beyond focal
plane
•
Potentially more sensitive
•
IR goes deeper into tissue
Disadvantages
•
Laser $$$
•
Samples with melanin
•
Samples with multiple
fluorescent labels
•
Slightly lower resolution
because of IR laser
Confocal Z-resolution an order of magnitude
worse than X-Y resolution
•
Confocal 3D data sets are not isotropic
•
Distortions along Z-axis
•
Higher N.A. not only improves X-Y resolution but also Z
•
Matching refractive index (
to avoid Z-axis artifacts
= speed of light in vacuum /speed in medium
Material
Refractive Index
Air
1.0003
Water
1.33
Glycerin
1.47
Immersion Oil
1.518
Glass
1.52
Diamond
2.42
Matching refractive index (
) and increasing
numerical aperture (N.A.)
to avoid Z-axis distortions
20x Dry
0.8 NA
40x water
1.2 NA
Matching refractive index (
) and increasing
numerical aperture (N.A.)
to avoid Z-axis distortions
40x Oil
1.3 NA
Matching refractive index (
) and increasing
numerical aperture (N.A.)
to avoid Z-axis distortions
20x Dry
1.52 NA corr
Matching refractive index (
) and increasing
numerical aperture (N.A.)
to avoid Z-axis distortions
Fluorescent proteins
•
Proteins from marine
invertebrates
•
Can be coded in genes
and made by the
organism
•
Now come in a variety
of colors
Green Fluorescent Protein (GFP)
•
First fluorescent protein
discovered and
developed for biological
use
•
Mutated for temp
stability, color and
turnover rate
•
Importance of
monomer vs dimer or
tetramer
Photoconvertible Proteins
•
Kaede, coral fluorescent protein,
tetramer
•
Dendra2, from soft coral, monomer
•
UV Laser (405 nm) to convert green to
red
•
ROI (Region Of Interest) allows precise
targeting
www.olympusfluoview.com
www.amalgaam.co.jp
Metric Prefixes
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:
1) Tbytes = Tera bytes = 10
12
Bytes
(storage capacity of computers)
2) Ghz = Gigahertz = 10
9
Hertz
(frequency)
3) M
= Megohm = Million Ohm
(resistance)
4) kW
= kilowattt
= 1000 Watt
(power)
¾ HP
5) hl
= hectoliter = Hundred liters
(volume of barrels)
6
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(
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)
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=
1
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7) cm
= centimeter
(length)
3/8”
8) mV
= millivolt
(voltage)
9) µA
= microampere
(current)
10) ng
= nanogram
(weight)
11) pf
= picofarad
(capacitance)
12) fl
= femtoliter
(volume)
Delve into the world of confocal microscopy with Lecture 07 of Principles of Modern Microscopy. Learn about optical sectioning, wide-field imaging, confocal laser scanning, and more to enhance fluorescence in microscopy. Understand the methods of optical sectioning such as deconvolution, multi-photon laser scanning, spinning disk systems, and structured illumination microscopy. Discover the relationship between diffraction, Airy disk, and point spread function for advanced imaging techniques.
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Presentation Transcript
Bi/BE 177: Principles of Modern Microscopy Lecture 07: Confocal Microscopy. Adding the Third Dimension Andres Collazo, Director Biological Imaging Facility Ke Ding, Graduate Student, TA Wan-Rong (Sandy) Wong, Graduate Student, TA
Lecture 7: Confocal Microscopy Optical Sectioning: adding the third dimension Wide-field Imaging Point Spread Function Deconvolution Confocal Laser Scanning Microscopy Confocal Aperture Optical aberrations Spinning disk confocal Two-photon Laser Scanning Microscopy Fluorescent proteins
Improve fluorescence with optical sectioning Wide-field microscopy Illuminating whole field of view Confocal microscopy Spot scanning Near-field microscopy For super-resolution TIRF Remember, typical compound microscope is not 3D, even though binocular
Overview of Optical sectioning Methods Deconvolution Point-Spread function (PSF) information is used to calculate light back to its origin Post processing of an image stack Confocal and Multi-photon Laser Scanning Microscopy Pinhole prevents out-of-focus light getting to the sensor(s) (PMT - Photomultiplier) Multi Photon does not require pinhole Spinning disk systems A large number of pinholes (used for excitation and emission) is used to prevent out-of- focus light getting to the camera Especially those using Nipkow disk and microlens Structured Illumination Microscopy (SIM) Light sheet fluorescence microscopy, also called selective/single plane illumination microscopy (SPIM)
Widefield imaging: entire field of view illuminated And projected onto a planar sensor
Relationship between diffraction, airy disk and point spread function Airy disk 2D Point spread function -3D Though often defined as the same that is not quite true
Point Spread Function is three dimensional Image of subdiffraction limit spot Subdiffraction limit spot https://micro.magnet.fsu.edu/primer/java/imageformation/depthoffield/index.html Thus, each spot in specimen will be blurred onto the sensor (Aperture and Missing Cone )
To reduce contribution of blurring to the image: Deconvolution Compute model of what might have generated the image Image blurred by PSF Compare and iterate Compute how model would be blurred by PSF Deconvolution depends on data from focal planes above and below focal plane being analyzed.
Image deconvolution Inputs: 3-D image stack 3-D PSF (bead image) Requires: Time Computer memory Artifacts? Algorithms so good now Note: z-axis blurring from the missing cone is minimized but not eliminated
Optical Sectioning even when 3D image stack is incomplete Deconvolution Confocal microscopy A A P Top: Macrophage - tubulin, actin & nucleus. Bottom: Imaginal disc -tubulin, -tubulin. Neural Gata-2 Promoter GFP-Transgenic Zebrafish; with Shuo Lin, UCLA
Optical Sectioning: Increased Contrast and Sharpness. Examples: Zebrafish images, Inner ear Zebrafish wide-field, optical section Confocal microscope Z-stack
How else to fill in the missing cone? Need more data in the Z-axis --> Confocal microscopy PMT Detector Detection Pinhole Confocal pinholes Excitation Laser Dichroic Beam Splitter Excitation Pinhole Objective Conjugate Focal Planes
Confocal Microscopy just a form of Fluorescence Microscopy www.olympusfluoview.com
Confocal Microscopy (Minsky, 1957) Yes that Marvin Minsky of MIT AI (Artificial Intelligence) lab fame. Three confocal places
Pinhole: Axial Filtering Identical Lens Focal Points
Aperture trims the PSF: increased resolution in XY plane How N.A. and air disk effects resolution But at a cost in brightness: Thinner section means less labeled material in image Aperture rejects some in focus light Subtle scattering or distortion rejects more light
Resolution, Signal and Pinhole Diameter Best Resolution Best Signal to Noise http://depts.washington.edu/keck/leica/pinhole.htm
Why does confocal add depth discrimination? Light projected on a single spot in the specimen Spatial filter in front of the detector Good: detection falls off by the distance from the focus squared Good: excitation falls off by the distance from the focus squared Bad: illumination of regions that are not used to generate an image Optical sectioning Combined, sensitivity falls off by (distance from the focus)4
But this arrangement generates an image of only one point in the specimen Only a single point is imaged at a time. Detector signal must be decoded by a computer to reconstruct image. Imaging point needs to be scanned somehow.
Scan Specimen Good: Microscope works on axis Best correction for optical aberrations Most uniform light collection efficiency Bad: Slow Sloshes specimen
Scan Microscope Head Good: Specimen doesn t move Microscope works on axis Best correction for optical aberrations Most uniform light collection efficiency Bad: Slow Optics can be more complicated
Scan Laser Good: Faster Specimen moves slowly less sloshing Bad: Very high requirements on objective Light collection may be non-uniform off-axis More complicated
Confocal Terminology LSCM Laser Scanning Confocal Microscopy CLSM Confocal Laser Scanning Microscopy CSLM Confocal Scanning Laser Microscopy LSM Laser Scanning Microscopy
Optical Aberrations: Imperfections in optical systems Spherical Chromatic (blue=shorter wavelength) Curvature of field
Spherical Aberration Zone of Confusion
Higher index of refraction results in shorter f Chromatic Aberration Lateral (magnification) Axial (focus shift) f Shift of focus i o Change in magnification
Lateral chromatic aberration - light misses aperture Detector
Curvature of field: Flat object does not project a flat image f i o Results in a port hole image: dimmer at edges
Optical Aberrations: Image dimmer with depth Image dimmer at edges Image resolution compromised N.A. = sin Can t fight losses with smaller NA Remember N.A. and image brightness Epifluorescence Brightness = fn (NA4 / magnification2) 10x 0.5 NA is 8 times brighter than 10x 0.3NA
Optical Aberrations: Image dimmer with depth Image dimmer at edges Image resolution compromised N.A. = sin Can t fight losses with smaller NA NA also has a major effect on image resolution Minimum resolvable distance dmin = 1.22 / (NA objective +NA condenser) Larger N.A. can collect higher order rays
Best way to deal with Aberrations is to have high performance objective High Numerical Aperture Water immersion for live cell imaging Correction for spherical aberrations Flat field correction Chromatically corrected over many different wavelengths Transmit UV and IR Two photon
How to scan the laser beam? Place galvanometer mirror at the telecentric point All light travels through the same zone Angle at which the light travels dictates the position in the specimen plane Not imaging but illumination conjugate plane. Telecentric Plane
How to scan the laser beam? Place galvanometer mirror at the telecentric point laser Modern closed-loop galvanometer-driven laser scanning mirror from Scanlab
Position is critical Place galvanometer mirror at the telecentric point laser If not at telecentric point, Spherical aberration results How can two mirrors be at the same point??
Scanners can introduce optical aberrations Place galvanometer mirror at the telecentric point laser If not at telecentric point, Spherical aberration results How can two mirrors be at the same point?? Optical relay (without aberration)
Problem: Optical aberrations from simple lens systems f i o
Simple pair of lenses can minimize problem (equal and opposite distortions) Focal Point Focal Point f
1:1 Image relay Focal Point f
Position is critical Place galvanometer mirror AT AT the telecentric point Optically two mirrors can be at the same point Optical relay (without aberration)
Limitations: Phototoxicity Sample is continuously exposed to light. Weaker signal within sample requires stronger excitation and causes more toxicity.
Limitations: Photobleaching Scanning causes repeated exposure above and below.
How else to do confocal microscopy? Confocal microscopes can be slow. Can we go faster?
Tandem spinning disk scanner EMCCD or CMOS Camera Laser Illumination through this side Detection through this side Alignment is critical Most of light hits mask not hole
Nipkow disk ~1% pass
Nipkow disk with microlenses >>1% pass Yokogawa
Nipkow disk with microlenses http://zeiss-campus.magnet.fsu.edu/tutorials/spinningdisk/yokogawa/index.html
Optical sectioning without an aperture? Two-Photon laser-scanning microscopy Pinhole aperture
